title: Arginine oscillation explains Na+ independence in the substrate/product antiporter CaiT.
abstract: Most secondary-active transporters transport their substrates using an electrochemical ion gradient. In contrast, the carnitine transporter (CaiT) is an ion-independent, l-carnitine/γ-butyrobetaine antiporter belonging to the betaine/carnitine/choline transporter family of secondary transporters. Recently determined crystal structures of CaiT from Escherichia coli and Proteus mirabilis revealed an inverted five-transmembrane-helix repeat similar to that in the amino acid/Na(+) symporter LeuT. The ion independence of CaiT makes it unique in this family. Here we show that mutations of arginine 262 (R262) make CaiT Na(+)-dependent. The transport activity of R262 mutants increased by 30-40% in the presence of a membrane potential, indicating substrate/Na(+) cotransport. Structural and biochemical characterization revealed that R262 plays a crucial role in substrate binding by stabilizing the partly unwound TM1' helix. Modeling CaiT from P. mirabilis in the outward-open and closed states on the corresponding structures of the related symporter BetP reveals alternating orientations of the buried R262 sidechain, which mimic sodium binding and unbinding in the Na(+)-coupled substrate symporters. We propose that a similar mechanism is operative in other Na(+)/H(+)-independent transporters, in which a positively charged amino acid replaces the cotransported cation. The oscillation of the R262 sidechain in CaiT indicates how a positive charge triggers the change between outward-open and inward-open conformations as a unifying critical step in LeuT-type transporters.
authors.affiliation: Department of Structural Biology, Max Planck Institute of Biophysics, 60438 Frankfurt am Main, Germany.
authors.forename: Sissy
authors.initials: S
authors.lastname: Kalayil
authors.forename: Sabrina
authors.initials: S
authors.lastname: Schulze
authors.forename: Werner
authors.initials: W
authors.lastname: Kühlbrandt
mesh_terms: D000595:Amino Acid Sequence; D017920:Antiporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport / Q000235:genetics; D002331:Carnitine / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011513:Proteus mirabilis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D001120:Arginine; D012964:Sodium; D002331:Carnitine
keywords: membrane protein structure; membrane transport mechanism; secondary-active transport; sodium-dependent transport; substrate/product antiport